The possibility of plant cell, tissue and/or organ culture--more commonly, and generically, referred to as plant tissue culture--was surmised as early as 1902 by G. Haberlandt. Although unsuccessful, he reasoned that hormonal regulation of cell division, growth and differentiation would be the key to successful organogenesis. Approximately a half century later, in 1956, Messrs. Skoog and Miller established that organogenesis was, in fact, regulated by quantitative interactions between growth factors, especially auxins and cytokinin in the culture medium. High auxin levels induce root initiation and repress shoot formation, whereas high cytokinin levels have the opposite effect. Once it was appreciated that an auxin and cytokinin balance is a general phenomenon critical to organogenesis, tissue culture technology became a commercial propagation tool rather than a mere academic exercise.
The two principle advantages of using tissue culture technology are that: (1) it permits rapid clonal propagation of plants with selected attributes and in large quantities; and (2) it permits recovery and propagation of specific pathogen-free plants i.e., plants free of specifically known and detectable fungi, bacteria, viruses, and viroids.
From a highly practical point of view tissue culture greatly reduces the vast greenhouse space heretofore required to house the parental stock inasmuch as microcuttings and plantlets are now readily available, year round, from parental stock cultures in micropropagation laboratories. Moreover, tissue culture production can be initiated at any time the appropriate plant explants are available, i.e., actively growing shoot tips, runner tips, etc.
Although specific procedures may well vary, most tissue culturists employ the same basic steps. Preparatory to the four stages of the actual tissue culture process, of course, one must first determine which part of the plant will be used. Because various plants may react differently, the specific part of the plant that can be most successfully employed for propagation by virtue of the tissue culture technology must be determined. For example, with ferns the runner tip is preferred, but other plants may culture more effectively from lateral buds, shoot tips, leaf parts or even sections of pollen, seeds or fruit. If a specific plant has been researched, the explant and cultural information will generally be available; otherwise, the lab propagator must develop that information on his own or have it developed through a research laboratory. Once determined, the appropriate plant parts, or "explants", are removed, their surfaces are disinfected, and they are inserted, according to the most rigorous aseptic techniques, into a previously prepared starter medium in a culture vessel. The vessel is then capped, or otherwise closed, to minimize contamination from the external environment.
Before continuing with a general background explanation of the heretofore employed tissue culture techniques, it should be appreciated that one must select a starter medium appropriate to the particular plant that is to be propagated. One exemplary starter medium that can be employed with a wide variety of plants is an inorganic salt mixture such as the well known Murashige and Skoog mixture which contains organic chemicals with the proper auxin/cytokinin balance for the particular plant being propagated by the tissue culture technique.
Such a mixture contains a variety of salts--viz., nitrates, sulfates, halides, potassium, boron and molybdenum--as well as iron, ethylene diamine tetraacetic acid, vitamins, sugar and the auxin/cytokinin balance. This mixture is added to distilled water and, though not necessarily, agar (a jelly-like substance made from seaweed) and brought to the desired pH by the addition of sodium hydroxide and/or hydrochloric acid. Thereafter, the mixture, if it contains agar, is heated and agitated until the agar is melted. While still hot the mixture is dispensed into tubes, or other culture vessels, which are then closed, as by stoppers, foil or suitable plastic wrap, and sterilized.
After the sterilized, starter medium cools it is ready for "Stage I" which is initiated by the aforedescribed insertion of the explants into the starter medium contained within the culture vessels. Throughout Stage I the closed vessels are maintained in an environment of controlled photoperiod, light intensity and temperature for a period of time appropriate to the particular plant. After about six weeks (the average time) the explant will have enlarged and may have formed a cluster of cells (termed a "callus"), and axillary shoots or adventitious shoots may be evident.
Stage II begins when those explants which are visibly free of contamination and which have established themselves in the starting medium of Stage I are subdivided and cultured in a multiplication, or nutrient, medium (this medium differs from the starter medium by employing a higher cytokinin to auxin ratio). At this stage the weaker explants, or propagules, and the unwanted "sports" can be eliminated. The subdivision and culturing process of Stage II can usually be repeated up to three or four times, but after that epigenetic changes can become an increasing problem with some plant types. During Stage II the propagules are not only very delicate but are also heterotrophic and receive all their nourishment from the medium itself; there is little or no photosynthesis occuring to provide food. Moreover, the propagules must be maintained in a sterile environment inasmuch as the sugar in the multiplication, or nutrient, medium provides a growth medium for unwanted micro-organisms, as well.
At the time of their last reculture and division the propagules enter Stage III. In Stage III the propagules are of sufficient number and of such size that they are ready for rooting and transfer to a pre-transplant, or hardening medium (this medium differs from the multiplication, or nutrient, medium, by employing a higher auxin to cytokinin ratio). This stage involves rooting the propagule in vitro, initiating the change from the heterotrophic stage (in which the cultured tissue derives nourishment solely from that added to the culture medium) to the autotrophic state (self nourishing by virtue of photosynthesis) and aclimating the resulting plantlet to be capable of survival in vivo.
After several weeks in Stage III the plantlets must be established in an in vivo medium, such as soil or a soil-like medium; this constitutes Stage IV. The transition which must be accomplished during Stage IV is perhaps the most drastic environmental change experienced during the tissue culture process. Specifically, the plantlet is subjected to lower humidity, higher light levels and for the first time it has been transplanted from an in vitro culture to an in vivo soil-like medium.
If the conditions are not carefully controlled, the plantlets may die, or exhibit reduced subsequent development. The soil-like medium must be prepared to be free of pathogens while providing sufficient soil irrigation and moisture to stimulate rapid root development. Moreover, high humidity, reduced light and moderate to warm temperatures must be initially maintained and gradually changed to the ambient environmental conditions of a greenhouse over several weeks to effect a satisfactory transition.
As should certainly be appreciated, the multiple steps involved in the tissue culture process are highly labor intensive. Not only must the tissues be repeatedly handled, the culture medium must be formulated differently from stage to stage.
The agar based media that is currently rather extensively employed in the tissue culture process requires a heating-cooling cycle in conjunction with the preparation of the culture media. Moreover, a high concentration of sugar is required in the agar medium to provide a nutrient to the heterotrophic propagules during root initiation. This necessitates washing the sugar off the plantlets immediately prior to their being transplanted into the Stage IV environment in order to minimize the likelihood of micro-organism development which would reduce plantlet survival in vivo.